The work of this Project is aimed at understanding and developing protein architectural design features that yield the specific dynamics and energy landscape within the Michaelis complex giving nse to enzymatic catalysis. The overall goal is to lay a foundation for the development and rational design of 'allosteric' effectors or active site inhibitors based on dynamics. This project will emphasize studies on lactate dehydrogenase (LDH), a hydride transfer enzyme essential in metabolism. It is used as a laboratory for our purposes, partly because the relevant dynamics can be studied quantitatively in unique detail, using our suite of very effective theoretical and experimental approaches, and associated with protein architecture. Other prototype enzymes, punne nucleoside phosphorylase (PNP) and dihydrofolate reductase (DHFR), will also be studied in collaboration with the other Projects. The goal of this research is to lay a foundation for the development and rational design of 'allosteric' effectors, that either up or down regulate enzymatic activity, or active site inhibitors based on dynamics. We have four specific aims: (1) examine the effects on the energy landscape of LDH's Michaelis complex in its relation to activity of specific mutant proteins, in response to osmolytes (for example TMAO and urea which are known to, respectively, increase and decrease the stability of folded proteins and hence, affect the energy landscape of an enzyme), and of so-called 'heavy and light' enzymes;'(2) probe how evolutionary pressure has affected the energy landscape of LDH to regulate activity by examining how adaptation to different thermal environments has occurred; (3) investigate the relationship of protein dynamics to allostery directly and work out how allosteric effectors affect the energy landscape of the protein system. All this provides a very robust set of experimental input into the theoretical studies of Project 4 (Schwartz). The small molecule allosteric effectors predicted by the Schwartz lab will be assessed; and (4) characterize functionally important enzyme motions within the Michaelis complex of PNP in direct collaboration with Projects 2 and 3.